therascreen PIK3CA RGQ PCR Kit

P190004 · QIAGEN GmbH · OWD · May 24, 2019 · Medical Genetics

Device Facts

• Record ID: P190004
• Device Name: therascreen PIK3CA RGQ PCR Kit
• Applicant: QIAGEN GmbH
• Product Code: OWD · Medical Genetics
• Decision Date: May 24, 2019
• Decision: APCB
• Device Class: Class 3

Intended Use

The therascreen PIK3CA RGQ PCR Kit is a real-time qualitative PCR test for the detection of 11 mutations in the phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) gene (Exon 7: C420R; Exon 9: E542K, E545A, E545D [1635G>T only], E545G, E545K, Q546E, Q546R; and Exon 20: H1047L, H1047R, H1047Y) using genomic DNA (gDNA) extracted from formalin-fixed, paraffin-embedded (FFPE) breast tumor tissue or circulating tumor DNA (ctDNA) from plasma derived from K₂EDTA anticoagulated peripheral whole blood taken from patients with breast cancer. The test is intended to aid clinicians in identifying breast cancer patients who may be eligible for treatment with PIQRAY® (alpelisib) based on a PIK3CA Mutation Detected result. Patients whose FFPE tissue or plasma specimen produce a positive therascreen PIK3CA RGQ PCR Kit test result for the presence of one or more PIK3CA mutations are eligible for treatment with PIQRAY (alpelisib). Patients whose plasma specimens produces a negative result using this test should be reflected to testing with FFPE tumor tissue for the presence of PIK3CA mutations. FFPE tumor specimens are processed using the QIAamp DSP DNA FFPE Tissue Kit for manual sample preparation. K₂EDTA anticoagulated whole peripheral venous blood plasma specimens are processed using the QIAamp DSP Circulating Nucleic Acid Kit for manual sample preparation. For both specimen types, the Rotor-Gene Q (RGQ) MDx (US) instrument is used for automated amplification and detection. The Kit is to be used by trained personnel in a professional laboratory environment.

Device Story

Device detects 11 PIK3CA mutations in breast cancer patients using ctDNA from K2EDTA plasma or FFPE tissue. Input: DNA extracted via QIAamp DSP Circulating Nucleic Acid Kit (plasma) or QIAamp DSP DNA FFPE Tissue Kit (tissue). Operation: Real-time PCR on Rotor-Gene Q MDx instrument using ARMS primers, LNA/TaqMan/MGB probes, and PCR clamps. Software (RGAM v2.1) performs automated amplification, detection, and result interpretation. Output: 'Mutation Detected' or 'No Mutation Detected'. Used in professional labs by trained personnel. Results aid clinicians in selecting patients for PIQRAY (alpelisib) therapy. Plasma-negative results require reflex testing with FFPE tissue to mitigate false-negative risk.

Clinical Evidence

Retrospective analysis of 543 evaluable patients from the SOLAR-1 clinical trial. Concordance between plasma and tissue testing showed PPA 54.6%, NPA 97.2%, OPA 71.5%. Clinical efficacy in plasma PIK3CA-positive patients showed 46% risk reduction in disease progression or death (HR=0.54, 95% CI: 0.33, 0.88) compared to placebo.

Technological Characteristics

Real-time PCR assay using ARMS primers, LNA/TaqMan/MGB probes, and PNA/phosphate-group PCR clamps. Instrument: Rotor-Gene Q MDx (centrifugal rotary thermal cycling). Connectivity: Standalone instrument. Software: RGAM v2.1 with Gamma MDx plug-in and assay-specific profile. Detection: FAM, HEX, ROX, CY5.5 fluorescent channels.

Indications for Use

Indicated for breast cancer patients to identify eligibility for PIQRAY (alpelisib) treatment via detection of 11 PIK3CA mutations in FFPE tumor tissue or plasma ctDNA. Plasma-negative patients require reflex testing with FFPE tissue.

Regulatory Classification

Identification

The somatic gene mutation detection system is an in vitro diagnostic nucleic acid amplification and real-time detection system for the identification of genetic mutations in DNA extracted from human tumor tissues.

Reference Devices

• therascreen PIK3CA RGQ PCR Kit (P190001)

Submission Summary (Full Text)

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SUMMARY OF SAFETY AND EFFECTIVENESS DATA (SSED)

I. GENERAL INFORMATION

Device Generic Name: Real-time PCR test

Device Trade Name: therascreen PIK3CA RGQ PCR Kit

Device Procode: OWD

Applicant's Name and Address: QIAGEN GmbH
QIAGEN Strasse 1
Hilden, 40724
Germany

Date(s) of Panel Recommendation: None

Premarket Approval Application (PMA) Number: P190004

Date of FDA Notice of Approval: May 24, 2019

The current PMA was submitted for an indication for the therascreen PIK3CA RGQ PCR Kit to identify 11 mutations in phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) gene (Exon 7: C420R; Exon 9: E542K, E545A, E545D [1635G>T only], E545G, E545K, Q546E, Q546R; and Exon 20: H1047L, H1047R, H1047Y) from circulating tumor DNA (ctDNA) isolated from K₂EDTA plasma from breast cancer patients who may be eligible for treatment with PIQRAY® (alpelisib).

Another PMA (P190001) for the same device using tissue specimens was also submitted for the qualitative detection of the same 11 mutations in the PIK3CA gene (Exon 7: C420R; Exon 9: E542K, E545A, E545D [1635G>T only], E545G, E545K, Q546E, Q546R; and Exon 20: H1047L, H1047R, H1047Y) in DNA derived from formalin-fixed, paraffin-embedded (FFPE) breast tumor tissue for the treatment with PIQRAY (alpelisib). P190001 was also approved on May 24, 2019 in conjunction with P190004 approval.

The summary of safety and effectiveness data (SSED) to support the indication is available on the Center for Devices and Radiological Health (CDRH) website.

II. INDICATIONS FOR USE

The therascreen PIK3CA RGQ PCR Kit is a real-time qualitative PCR test for the detection of 11 mutations in the phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) gene (Exon 7: C420R; Exon 9: E542K, E545A, E545D [1635G>T only], E545G, E545K, Q546E, Q546R; and Exon 20: H1047L, H1047R, H1047Y) using genomic DNA (gDNA) extracted from formalin-fixed, paraffin-embedded (FFPE)

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breast tumor tissue or circulating tumor DNA (ctDNA) from plasma derived from K₂EDTA anticoagulated peripheral whole blood taken from patients with breast cancer.

The test is intended to aid clinicians in identifying breast cancer patients who may be eligible for treatment with PIQRAY® (alpelisib) based on a PIK3CA Mutation Detected result. Patients whose FFPE tissue or plasma specimen produce a positive therascreen PIK3CA RGQ PCR Kit test result for the presence of one or more PIK3CA mutations are eligible for treatment with PIQRAY (alpelisib). Patients whose plasma specimens produces a negative result using this test should be reflected to testing with FFPE tumor tissue for the presence of PIK3CA mutations.

FFPE tumor specimens are processed using the QIAamp DSP DNA FFPE Tissue Kit for manual sample preparation. K₂EDTA anticoagulated whole peripheral venous blood plasma specimens are processed using the QIAamp DSP Circulating Nucleic Acid Kit for manual sample preparation. For both specimen types, the Rotor-Gene Q (RGQ) MDx (US) instrument is used for automated amplification and detection. The Kit is to be used by trained personnel in a professional laboratory environment.

## III. CONTRAINDICATIONS

There are no known contraindications.

## IV. WARNINGS AND PRECAUTIONS

The warnings and precautions can be found in the therascreen PIK3CA RGQ PCR Kit labeling.

## V. DEVICE DESCRIPTION

The following components comprise the overall device for testing plasma specimens:

- QIAGEN QIAamp® DSP Circulating Nucleic Acid Kit
- QIAGEN therascreen PIK3CA RGQ PCR Kit
- QIAGEN Rotor-Gene Q MDx Instrument (RGQ)
- Automated data analysis and results interpretation using:
- Rotor-Gene AssayManager® (RGAM) software version 2.1
- Rotor-Gene AssayManager Gamma MDx plug-in version 1.0.0
- therascreen_PIK3CA_Plasma Assay Profile v1.0.1

## Specimen Preparation – ctDNA

Peripheral whole blood is collected from breast cancer patients using blood collection tubes containing di-potassium ethylenediamine tetraacetic acid (K₂EDTA) as an anticoagulant. The tubes are then centrifuged to pellet the cellular components of the blood before the plasma fraction is transferred to a fresh tube. ctDNA is manually extracted and purified

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from the blood plasma using the QIAamp DSP Circulating Nucleic Acid Kit. QIAamp Mini columns can bind fragmented nucleic acids that are as short as 20 base pairs (bp), but the yield depends on the sample volume and the concentration of circulating nucleic acids in the sample (typically 1–100 ng/mL circulating nucleic acids in plasma). All assays in the therascreen PIK3CA RGQ PCR Kit are designed to amplify short DNA fragments with amplicons ranging from 75 bp to 126 bp, however, the therascreen PIK3CA RGQ PCR Kit will not work with heavily fragmented DNA. If a sample fails the DNA sample assessment (Control Ct value is >31.68), the purification process should be repeated with new samples.

The ctDNA purification process involves the following steps:

- Samples are lysed under denaturing conditions with proteinase K for 30 minutes at 60°C.
- The sample is passed through a silica-based membrane so that ctDNA binds to the membrane and contaminants are removed.
- The membrane is washed with buffers (using the QIAvac 24 Plus vacuum manifold to increase flow rates through the membrane).
- Purified ctDNA is eluted from the membrane using buffer.

Extracted ctDNA is ready for testing with the therascreen PIK3CA RGQ PCR Kit or can be stored at –30 to –15°C for up to 4 week before use.

The QIAamp DSP Circulating Nucleic Acid Kit provides reagents that enable standardized manual purification of cfDNA from blood plasma using a pre-defined protocol. The DNA extraction process should be followed as per kit instruction with the following therascreen PIK3CA RGQ PCR Kit custom steps:

- Instructions for sample volumes of 2 mL of plasma must be followed. Where 2 mL is not available, the volume must be adjusted to 2 mL using Phosphate-buffered Saline (PBS).

Note: The extracted DNA should be within working control Ct range (24.69 ≥ and ≤ 31.68) for the sample to be valid.

- Proteinase K volume must be 250 μL.
- Purified ctDNA must be eluted in 70 μL of elution buffer (Buffer AVE) from the QIAamp DSP Circulating Nucleic Acid Kit.

## PCR Amplification and Detection

The QIAGEN therascreen PIK3CA RGQ PCR Kit contains reagents for the detection of 11 individual mutations in exons 7, 9, and 20 of the phosphoinositide-3-kinase, catalytic, alpha (PIK3CA) gene, and a region in exon 15 of the PIK3CA gene used as a Control Reaction. The detection of the 11 individual mutations and region in exon 15 of the PIK3CA gene is achieved using six reaction mixes listed below:

Table 1: PIK3CA Mutations Detected by the therascreen PIK3CA RGQ PCR Kit

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| Reaction Mix | PIK3CA Mutation | Base change | Exon | COSMIC ID1 |
| --- | --- | --- | --- | --- |
| 1 | Control Reaction | N/A | 15 | N/A |
| 2 | E542K | 1624 G>A | 9 | 760 |
| 3 | E545K | 1633 G>A | | 763 |
| | E545D | 1635 G>T | 9 | 765 |
| | E545G | 1634 A>G | | 764 |
| 4 | E545A | 1634 A>C | 9 | 12458 |
| | H1047Y | 3139 C>T | 20 | 774 |
| | Q546R | 1637 A>G | 9 | 12459 |
| 5 | C420R | 1258 T>C | 9 | 757 |
| | Q546E | 1636 C>G | 7 | 6147 |
| 6 | H1047L | 3140 A>T | 20 | 776 |
| | H1047R | 3140 A>G | | 775 |

$^{1}$ COSMIC IDs taken from the Catalogue of Somatic Mutations in Cancer: https://cancer.sanger.ac.uk/cosmic

The therascreen PIK3CA RGQ PCR Kit uses real-time PCR with the following technologies for PIK3CA sequence related amplification and detection: ARMS® Primers, LNA® Probes, TaqMan® Probes, Minor Groove Binding (MGB™) Probes, and PCR Clamps for Wild Type (WT) and pseudogene sequences.

PCR uses forward and reverse primers to hybridize to a specific DNA sequence to amplify it. The ARMS technique is based on the use of mutation sequence-specific PCR primers that allow amplification of test DNA only when the target allele is contained within the sample.

In addition to the primers, dye-linked oligonucleotides (i.e., probes; e.g., LNA, TaqMan and MGB) are contained in the reaction mixes. The probes, which are labeled with a $5^{\prime}$ reporter dye (carboxyfluorescein $\left[\mathrm{FAM}^{\mathrm{TM}}\right]$ ) and a downstream, $3^{\prime}$ dye-free quencher (i.e., BHQ1) which quenches the fluorescence of the reporter dye, also hybridize to the target sequence between the primers. When a probe is intact, the proximity of the reporter dye to the quencher results in suppression of the reporter fluorescence primarily by Förster-type energy transfer.

PCR clamp technology allows selective amplification of the mutant sequence. PCR clamps matched to WT or pseudogene sequence bind to the template and prevent amplification by interference with primer elongation. There are two types of PCR clamps used within the therascreen PIK3CA RGQ PCR Kit; peptide nucleic acids (PNAs) and $3^{\prime}$ phosphate groups.

During PCR, forward and reverse primers and a probe bind to the target sequence. DNA polymerase extends the primers and the $5^{\prime}$ to $3^{\prime}$ exonuclease activity of the enzyme cleaves the probe between the reporter and the quencher leading to an increase in detectable reporter fluorescence. This process occurs in every PCR cycle. The increase in fluorescence is directly proportional to the target amplification during PCR.

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The probes used in the mutation specific reaction mixes are labeled with carboxyfluorescein (FAM), hexachloro-fluorescein (HEX) and Cyanine (CY5.5) fluorescent reporter dyes, each with a distinct absorption and emission profile. The probe used in the Internal Control Reaction is labeled with Rhodamine (ROX).

FAM, HEX, ROX and CY5.5 absorb and fluoresce at different wavelengths:

- FAM: 6-carboxyfluorescein: A fluorophore that excites at a wavelength of $495\mathrm{nm}$ and emits at a wavelength of $520\mathrm{nm}$ . This fluoresces in the green RGQ channel.
- HEX: Hexachloro-fluorescein: A fluorophore that excites at a wavelength of $535\mathrm{nm}$ and emits at a wavelength of $556\mathrm{nm}$ . This fluoresces in the yellow RGQ channel.
- ROX: A fluorophore of the Rhodamine family that excites at $578\mathrm{nm}$ and emits at $604\mathrm{nm}$ . This fluoresces in the orange RGQ channel.
- CY5.5: A fluorophore of the Cyanine family that excites at $675\mathrm{nm}$ and emits at $694\mathrm{nm}$ . This fluoresces in the crimson RGQ channel.

The Control Reaction Mix contains a forward and reverse primer and labeled probe (detected in the Green Channel) to amplify a short sequence of exon 15 of the PIK3CA gene. The Control Reaction is used to determine if an appropriate level of amplifiable DNA is present in the sample and is a factor in the analytical calculations that determine mutation status. All samples must be tested with the Control Reaction Mix (Tube 1) to ensure that they give Ct values within a specified range to ensure that there is enough amplifiable DNA to proceed with analysis, but not so much as to overload the assay. The Control Reaction determines whether the quality and quantity of DNA is sufficient for the working range of the assay. The interpretation of the results obtained from the Control Reaction Ct is presented below in Table 2. Any samples that do not give Ct values within this range are invalidated by the RGAM software.

Table 2: Control Reaction Working Range

| Control Ct value | Interpretation | Action |
| --- | --- | --- |
| >31.68 | Quantity of amplifiable ctDNA is not sufficient for mutation analysis | Additional samples should be extracted and tested |
| <24.69 | Quantity of amplifiable ctDNA is too high for mutation analysis | Dilute with the sample diluent water supplied in the kit |
| ≥24.69 and ≤31.68 | Quantity of amplifiable ctDNA is suitable for mutation analysis | No action required, sample is suitable |

The PCR cycling parameters used for assessing the DNA sample with the control reaction mix are the same run parameters for mutation analysis using the mutation assays. The PCR cycling parameters used for assessing the DNA sample are:

- Hold at $95^{\circ}\mathrm{C}$ for 15 minutes to activate the Taq polymerase;

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- PCR for 45 cycles of 95°C for 30 seconds, to denature, and 60°C for 1 minute, to anneal/extend.

If the control assay Ct falls within range, then the sample is analyzed for the presence of the mutation by analyzing the values obtained in the mutation channels and completing the ΔCt calculation. If the control assay Ct is not within range the sample is considered invalid and any results obtained may not be used to make a mutation status evaluation. This assessment is performed automatically by the RGAM software and associated plug-in and assay profile.

## Test Controls

The therascreen PIK3CA RGQ PCR Kit contains three controls: An Internal Control (IC), a Positive Control (PC) and a No Template Control (NTC), which have been designed to detect fault conditions.

**Internal Control (IC)**: Each PIK3CA reaction mix contains reagents (unlabeled primers, probe and oligonucleotide template) for an IC reaction designed to detect failure of the reaction, e.g. due to set up error, and confirms successful PCR reactions in every tube.

**No Template Control (NTC)**: An NTC test contains nuclease-free water and is required in each RGQ run. The NTC serves as a control to assess potential contamination during assay set up.

**Positive Control (PC)**: A PC test is required in each RGQ run. The PC Tube comprises a mixture of plasmids representing one mutation for each of the mutation assays and the Control Assay. Detection of the targets within their acceptable ranges confirms the proper functioning of each of the reaction mixes in the kit.

## Instrument(s) and Software

The therascreen PIK3CA RGQ PCR Kit is designed to be used with the RGQ instrument, which is a real-time PCR analyzer designed for rapid thermal cycling and real-time detection of PCR assays.

The RGQ incorporates a centrifugal rotary design for thermal cycling where a rotor, containing each tube, spins in a chamber of moving air, keeping all samples at a uniform temperature. Samples are heated and cooled in a low-mass-air oven according to a software-determined cycle that initiates the different phases of the PCR cycle. In the RGQ, fluorophores are excited from the bottom of the sample chamber by a light-emitting diode. Energy is transmitted through the thin wall at the bottom of each PCR tube. Emitted fluorescence passes through the emission filters on the side of the chamber and is detected by a photomultiplier tube. Detection is performed as each tube aligns with the detection optics; tubes spin pass the excitation / emission optics every 150 milliseconds. The fluorescence signals indicate the progress of the PCR reactions. The Rotor-Gene Q MDx has

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six channels (six excitation sources and six detection filters). Four of these channels; green, yellow, crimson and orange, are used with the therascreen PIK3CA RGQ PCR Kit.

Cycling parameters, data analysis and results interpretation for the therascreen PIK3CA RGQ PCR Kit are performed by the RGAM version 2.1 Software, Gamma MDx plug-in version 1.0.0 and therascreen_PIK3CA_Plasma Assay Profile v1.0.1. Therefore, no manual analysis is required.

The RGAM Software is a core software which provides general functionality including: PCR run set up, cycler control and management of experiment data, results, assay profiles and system configuration.

The Gamma MDx Plug-in extends the functionality of RGAM by providing cycle threshold (Ct) value calculation, data analysis and normalization features.

Assay specific functionality, for example cycling conditions, thresholds and analysis cut offs, and control ranges, is implemented by the therascreen_PIK3CA_Plasma Assay Profile.

The RGAM software, plug-in and associated assay profile ensure that a user interface with restricted user options is displayed to the user and contains all the information required for automatic real-time PCR analysis including time and temperature profiles, data quality controls, and data analysis algorithms. The software suite also allows printing of test reports and creates result files in the software's file system.

In addition, the RGAM software, plug-in and associated assay profile perform a quality check using Automatic Data Scan (AUDAS) that focuses on parameters of the respective fluorescence curves from which Ct values will be determined.

The AUDAS check is mainly intended to identify problems that occur during the real-time PCR amplification that potentially generate nontypical curve shapes due to saturation, noise, spikes, baseline dips, sloping curves related to the real-time PCR instrument parameters or due to a problem linked to the assay itself. The samples with curves in such situations are automatically invalidated to avoid generating misleading results.

## Interpretation of Results

The first cycle at which the instrument can distinguish the amplification generated fluorescence as being above the background signal is called the Ct. The RGAM software interpolates fluorescence signals between any two recorded values. Ct values can therefore be any number (not limited to integers) within the range of 0 to 45.

Ct values generated by the Control and Mutation Reactions indicate the quantity of assay specific input DNA and are indirectly proportional to the input DNA levels. Low Ct values indicate higher input DNA levels and high Ct values indicate lower input DNA levels. The interpretation of the results obtained from the control reaction Ct is presented in Table 2.

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Validity of controls and samples are determined based on the Ct values generated during a run.

# Run Validity Criteria

For a therascreen PIK3CA RGQ PCR Kit run to be accepted as valid, the RGAM software, plug-in and associated assay profile require run data for the PC and NTC, to meet specified criteria. Each test run performed with the therascreen PIK3CA RGQ PCR Kit must meet all the validity criteria listed below (Table 3).

Table 3:Run, Sample Validity and Call Criteria

| Sample | Reaction Mix | Target | RGQ Channel | Ct Range |
| --- | --- | --- | --- | --- |
| Positive Control | 1 and 5 | Control | FAM | 23.39 – 32.39 |
| | 2 | E542K | FAM | 22.42 – 31.42 |
| | 3 | E545D | HEX | 23.78 – 32.78 |
| | 3 | E545G | CY5.5 | 22.61 – 31.61 |
| | 3 | E545K | FAM | 24.41 – 33.41 |
| | 4 | E545A | FAM | 22.5 – 31.5 |
| | 4 | H1047Y | HEX | 26.57 – 35.57 |
| | 4 and 5 | Q546R | CY5.5 | 24.04 – 33.04 |
| | 5 | C420R | HEX | 23.31 – 34.31 |
| | 1 and 5 | Q546E | FAM | 24.72 – 35.72 |
| | 6 | H1047L | CY5.5 | 24.02 – 33.02 |
| | 6 | H1047R | HEX | 23.33 – 32.33 |
| NTC | All | All 6 reaction mixes | FAM | Has no value |
| IC | NTC | IC | ROX | 25.52 – 36.51 |
| | Test Sample | IC | ROX | 25.51 – 36.51 |
| | Positive Control | IC | ROX | 25.51 – 36.51 |
| Test Sample | 1 | Control | FAM | 23.39 – 32.39 |

If a test run fails any of the validity criteria, the RGAM software displays the corresponding validity rule related to the failed control but does not provide test results for samples on the RGAM report. If all run validity criteria are met, the RGAM generates a report that confirms the respective controls validity and then displays the sample results.

The individual sample results in each test run are accepted as valid, if the RGAM software obtains Ct values for the PIK3CA assay. If a sample fails to generate a Ct value for any PIK3CA mutant channel, then the RGAM software checks the Ct value obtained in the IC channel to ensure the qPCR reaction validity. If the RGAM fails to detect a signal within the validity criteria range in the IC, the sample is reported as invalid and no PIK3CA mutation status results for that sample are reported. For a therascreen PIK3CA RGQ PCR Kit run to be accepted as valid, the RGAM software, plug-in and associated assay profile require run data for the PC and NTC, to meet specified criteria.

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# Sample Validity Criteria and Control Assay Working Range

All samples must be tested with the Control Reaction mix to ensure that they give a Ct value within a specified range. This range is set to ensure that there is sufficient amplifiable DNA to proceed with analysis, but not so much as to overload the assay. The *therascreen* PIK3CA RGQ PCR Kit has been verified to work within a specific range (i.e. upper and lower Control Reaction Ct values) and any samples that do not give Ct values within this range are invalidated by the RGAM software, plug-in and associated assay profile.

# Determination of Sample Status

If the Control Assay Ct falls within range, then the sample is analyzed for the presence of the mutation. The difference in Ct values ($\Delta$Ct) between the Control Reaction and the mutation-specific reaction (mutation assay) is a qualitative measure of PIK3CA mutation status and is calculated as:

$$
\Delta \mathrm{Ct} = [\text{Mutation Reaction Ct value}] - [\text{Control Reaction Ct value}]
$$

Samples are classified as mutation positive if they give a $\Delta$Ct less than or equal to the cut-off $\Delta$Ct value for that assay. Above this value, the sample may either contain less than the percentage of mutation that can be detected by the *therascreen* PIK3CA RGQ PCR Kit (beyond the limit of detection of the assays), or the sample is mutation negative, both of which would be reported as “No Mutation Detected”.

The Mutation Assay $\Delta$Ct Cut-offs are shown below in Table 4.

Table 4: Cut-off Values for Each Mutation in Each Assay

| Reaction Mix | Mutation | Ct cut-off values |
| --- | --- | --- |
| Tube 2 | E542K | ≤ 4.80 |
| Tube 3 | E545K | ≤ 10.00 |
| | E545D | ≤ 7.00 |
| | E545G | ≤ 9.50 |
| Tube 4 | E545A | ≤ 10.00 |
| | H1047Y | ≤ 6.20 |
| | Q546R | ≤7.00 |
| Tube 5 | Q546E | ≤10.00 |

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| Reaction Mix | Mutation | Ct cut-off values |
| --- | --- | --- |
| | C420R | ≤6.00 |
| Tube 6 | H1047R | ≤9.00 |
| | H1047L | ≤10.00 |

On the RGAM report each sample is assigned with a status as follows:

Invalid:
- If one of the AUDAS checks failed
- or if one of the run control criteria failed
- or if the sample IC failed
- or if the Control Assay Ct was outside of the acceptance range

PIK3CA Mutation Detected:
- If all AUDAS checks passed
- and if all run control criteria were met
- and if the sample IC was within the defined acceptance range
- and if the Control Assay Ct was within the acceptable range
- and if any PIK3CA mutant signals were equal to or below the predefined $\Delta \mathrm{Ct}$ cut-off

No Mutation Detected:
- If all AUDAS checks passed
- and if all run control criteria were met
- and if the sample IC was within the defined acceptance range
- and if the Control Assay Ct was within the acceptable range
- and if all mutant signals were above the predefined $\Delta \mathrm{Ct}$ cut-off

VI. ALTERNATIVE PRACTICES AND PROCEDURES

There are no other FDA cleared or approved alternatives for the testing of plasma (or formalin-fixed, paraffin-embedded [FFPE] breast tumor tissue) for PIK3CA mutation status in the selection of patients who may be eligible for treatment with PIQRAY® (alpelisib).

VII. MARKETING HISTORY

The therascreen PIK3CA RGQ PCR Kit has not been marketed in the United States or any foreign country.

VIII. POTENTIAL ADVERSE EFFECTS OF THE DEVICE ON HEALTH

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Failure of the device to perform appropriately, or failure to correctly interpret test results may lead to incorrect PIK3CA mutation results, which could impact patient treatment decisions. A false positive test result may lead to inappropriate treatment and adverse effects associated with treatment with a targeted PIK3CA inhibitor rather than standard of care treatments. A false negative test result may prevent a patient from receiving alpelisib with potential to benefit from a targeted therapy.

For the specific adverse events that occurred in the clinical study, please see Section X below.

## IX. SUMMARY OF NONCLINICAL STUDIES

### A. Laboratory Studies

The specific performance characteristics of the therascreen PIK3CA RGQ PCR Kit were determined by studies using three types of test samples:

- Procured clinical plasma samples obtained from patients with advanced breast cancer.
- Contrived plasma samples comprising Healthy Donor (HD) EDTA plasma spiked with fragmented cell line DNA to mimic clinical plasma samples.
- Fragmented genomic DNA (gDNA) from cell line samples

Due to volume constraints of clinical plasma samples and due to the rarity of most of the mutations, contrived samples were utilized for some non-clinical studies. A contrived sample characterization study was conducted to demonstrate comparable performance of sheared cell line DNA diluted in HD plasma as compared to K₂EDTA plasma from clinical samples. Clinical specimens were used to confirm the estimated limit of detection (LoD), analytical accuracy and stability studies. Testing was performed using the final version of the therascreen PIK3CA RGQ PCR Kit, following established protocols. DNA was extracted and tested in accordance with the instructions for use.

### 1. Correlation with Reference Method

To demonstrate the analytical accuracy of the therascreen PIK3CA RGQ PCR Kit, results were compared with a validated Next Generation Sequencing (NGS)-based method using banked DNA from clinical plasma samples.

Plasma specimens were collected at baseline, prior to initiation of study treatment, from patients with breast cancer in the Novartis clinical study CBYL719C2301 (SOLAR-1, see section X). Samples were obtained from 554 of 572 total randomized patients enrolled into the SOLAR-1 clinical study. Eighteen (18) patients had no plasma samples available for testing. Of the 554, there were 552 samples with residual DNA available for use in this accuracy study. Of the 552 samples available, 5 samples were excluded from this analysis as the samples were collected after initiation of therapy, and an additional 5 did not have sufficient DNA for NGS analysis. The level of concordance

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between the therascreen PIK3CA RGQ PCR Kit and the validated NGS method was demonstrated by comparing the results obtained from both methods.

Specimens were processed and tested by the therascreen PIK3CA RGQ PCR Kit according to the final product labeling. Invalid and indeterminate samples were retested according to the clinical test site protocol.

The data from these samples were used to determine the level of concordance between the two methods and the overall percent agreement (OPA), positive percent agreement (PPA), and negative percent agreement (NPA) was reported with the corresponding two-sided Clopper-Pearson exact $95\%$ confidence limits (Table 5).

Table 5: Agreement in Overall Mutation Status between therascreen PIK3CA RGQ PCR Kit and NGS

| Measure Of Agreement | Frequencies | Percent Agreement | Clopper-Pearson (Exact) Binomial Lower Two-sided 95% Confidence Limit | Clopper-Pearson (Exact) Binomial Upper Two-sided 95% Confidence Limit |
| --- | --- | --- | --- | --- |
| Overall Percent | 504/542 | 92.99 | 90.50 | 94.99 |
| Positive Percent | 149/153 | 97.39 | 93.44 | 99.28 |
| Negative Percent | 355/389 | 91.26 | 88.00 | 93.87 |

The data generated in this accuracy study has demonstrated that the observed PPA was $97.39\%$ . The observed NPA was $91.26\%$ .

In addition, the agreement was evaluated at the mutation level rather than at sample level. The results are shown in the table below (Table 6).

Table 6: PIK3CA Specific mutation status against NGS specific mutation status

| Frequency | | Next Generation Sequencing Mutation Status | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| PIK3CA Plasma KitMutation Status | C420R | E542K | E542K, H1047R | E545G | E545K | H1047L | H1047R | Negative | Total |
| C420R | 2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 2 |
| E542K | 0 | 19 | 0 | 0 | 0 | 0 | 0 | 2 | 21 |
| E542K,H1047R | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 1 |
| E545G | 0 | 0 | 0 | 2 | 0 | 0 | 0 | 1 | 3 |

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| E545K | 0 | 0 | 0 | 0 | 38 | 0 | 0 | 8 | 46 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| H1047L | 0 | 0 | 0 | 0 | 0 | 5 | 0 | 5 | 10 |
| H1047R | 0 | 0 | 0 | 0 | 0 | 0 | 82 | 18 | 100 |
| Negative | 0 | 2 | 0 | 0 | 0 | 0 | 2 | 355 | 359 |
| Total | 2 | 21 | 1 | 2 | 38 | 5 | 84 | 389 | 542 |

In this study 38 discordant results were observed out of 542 samples. Of these 38 samples, 4 were classified as mutation detected by the NGS method but mutation not detected by the therascreen PIK3CA RGQ PCR Kit using plasma (Plasma negative/NGS positive), and 34 were therascreen PIK3CA plasma positive but NGS plasma negative, including 29 therascreen PIK3CA tissue positive and 5 therascreen PIK3CA tissue negative [(i.e., out of 34, 29 were (PIK3CA Tissue+, Plasma+); and 5 were (PIK3CA Tissue-, Plasma +)].

There were five (5) mutations (E545A, E545D, Q546E, Q546R and H1047Y) out of 11 PIK3CA mutations that were not detected when plasma samples were tested by the therascreen PIK3CA RGQ PCR Kit and by the NGS method. These samples tested positive when the corresponding tissue samples were tested using the therascreen PIK3CA RGQ PCR Kit.

## 2. Contrived Sample Functional Characterization

The objective of this study was to determine whether the contrived samples consisting of fragmented PIK3CA mutation cell-line DNA spiked into Healthy Donor (HD) plasma (contrived plasma samples) are equivalent to clinical PIK3CA mutation positive plasma samples derived from breast cancer patients.

The three most prevalent PIK3CA mutations, E545K, E542K and H1047R, were used to assess the performance of the kit using plasma specimens. Equivalency was assessed by comparison of "positivity rate" (mutation detected vs. mutation not detected) for the samples at the limit of detection (LoD). Fragmented DNA was spiked in from stocks at 50% mutation allele frequency (MAF). The DNA eluates were normalized to a control assay Ct of 30 in order to provide a challenging sample near the upper limit of the control working range. Four different mutant allele frequency (MAF%) levels (LoD to 0.35x LoD) were prepared for each mutation.

The prepared samples at different MAF levels were then tested with the control and corresponding mutation assays of the therascreen PIK3CA RGQ PCR Kit and subjected to statistical analysis of hit rate at the LoD MAF level. Based on these analyses, the results (Table 7) demonstrate that the performance of the therascreen PIK3CA RGQ PCR Kit is equivalent for both contrived plasma samples and clinical plasma samples. The 1X LoD values for the corresponding mutants are in bold in Table 7.

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Table 7: Results of Contrived Sample Characterization Study

| Grouping Variable(s) | | | Proportion | | Two-Sided 95% | |
| --- | --- | --- | --- | --- | --- | --- |
| Mutation | Sample Type | MAF[ | Fraction | Percentage | Lower | Upper |
| E545K | Clinical | 0.86 | 17 / 24 | 70.83% | 48.91% | 87.38% |
| | | 1.22 | 23 / 24 | 95.83% | 78.88% | 99.89% |
| | | 1.72 | 24 / 24 | 100.00% | 85.75% | 100.00% |
| | | 2.42 | 24 / 24 | 100.00% | 85.75% | 100.00% |
| | Contrived | 0.86 | 22 / 24 | 91.67% | 73.00% | 98.97% |
| | | 1.22 | 23 / 24 | 95.83% | 78.88% | 99.89% |
| | | 1.72 | 24 / 24 | 100.00% | 85.75% | 100.00% |
| | | 2.42 | 24 / 24 | 100.00 | 85.75 | 100.00% |
| H1047R | Clinical | 0.71 | 17 / 24 | 70.83% | 48.91 | 87.38% |
| | | 1 | 22 / 24 | 91.67% | 73.00 | 98.97% |
| | | 1.4 | 23 / 24 | 95.83% | 78.88 | 99.89% |
| | | 1.98 | 24 / 24 | 100.00 | 85.75 | 100.00% |
| | Contrived | 0.71 | 19 / 24 | 79.17% | 57.85 | 92.87% |
| | | 1 | 24 / 24 | 100.00 | 85.75 | 100.00% |
| | | 1.4 | 24 / 24 | 100.00 | 85.75 | 100.00% |
| | | 1.98 | 24 / 24 | 100.00 | 85.75 | 100.00% |
| E542K | Clinical | 1.804 | 11 / 24 | 45.83% | 25.55 | 67.18% |
| | | 2.544 | 15 / 24 | 62.50% | 40.59 | 81.20% |
| | | 3.587 | 23 / 24 | 95.83% | 78.88 | 99.89% |
| | | 5.058 | 24 / 24 | 100.00 | 85.75 | 100.00% |
| | Contrived | 1.804 | 11 / 24 | 45.83% | 25.55 | 67.18% |
| | | 2.544 | 19 / 24 | 79.17% | 57.85 | 92.87% |
| | | 3.587 | 22 / 24 | 91.67% | 73.00 | 98.97% |
| | | 5.058 | 24 / 24 | 100.00 | 85.75 | 100.00% |

# 3. Analytical Sensitivity

# (i) Limit of Blank (LoB)

The LoB is defined in CLSI guideline EP17-A2 as "the highest measurement result that is likely to be observed (with a stated probability) for a blank sample". For the therascreen PIK3CA RGQ PCR Kit this is the data point that corresponds to the upper $95\%$ percentile in the blank samples.

The LoB was confirmed by testing 60 unique HD plasma samples. The 60 samples were spiked with serially diluted fragmented Wild Type PIK3CA DNA at six DNA input levels to produce control assay Ct values spanning the therascreen PIK3CA RGQ PCR Kit (for plasma specimens) control assay working range (24.69 - 31.68). For each sample one replicate was tested with each of the three kit lots to give a total

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of 180 data points per assay. The lowest value of the three LoB estimates (one from each therascreen PIK3CA RGQ PCR Kit lot) was determined to be the LoB value.

The LoB values for each of the mutation assays (in terms of $\Delta \mathrm{Ct}$ ) detected by the therascreen PIK3CA RGQ PCR Kit were verified to be above the $\Delta \mathrm{Ct}$ cut-off values determined for each of the assays as summarized in Table 8. The false positive call rates were also calculated as summarized in Table 8.

# (ii) $\Delta C t$ Analytical Cut-off Validation

For the $\Delta C t$ cut-off verification the following acceptance criteria were set: For each mutation (i.e. each tube and each channel), using only the WT samples and using the final assay cut-off value in terms of $\Delta C t$ , the estimated false positive rate must be less than $3 \%$ . Using the WT sample data, the false positive fraction was calculated using the cutoffs set in development. As the proportion of false positive calls in this study was found to be 2/180 $(1.11\%)$ across all 11 mutation assays, meeting the acceptance criteria, these cut-offs were used as the final assay cutoffs.

Table 8: LoB and false positive rate for each Mutation Assay

| Reaction Mix | Assay | LoB (ΔCt) | False positive rate (%) |
| --- | --- | --- | --- |
| Tube 2 | E542K | 8.32 | 0.00% |
| Tube 3 | E545K | 15.74 | 0.00% |
| | E545D | 9.13 | 0.00% |
| | E545G | 13.39 | 0.00% |
| Tube 4 | E545A | 15.82 | 0.00% |
| | H1047Y | 9.89 | 0.00% |
| | Q546R | 10.19 | 0.56% |
| Tube 5 | Q546E | 15.82 | 0.00% |
| | C420R | 11.15 | 0.00% |
| Tube 6 | H1047R | 11.93 | 0.00% |
| | H1047L | 15.55 | 0.56% |

# (iii) Limit of Detection (LoD)

The design of the study was guided by the CLSI guideline EP17-A2. The LoD for each mutation assay in the therascreen PIK3CA RGQ PCR Kit was estimated using contrived plasma samples. For the therascreen PIK3CA RGQ PCR Kot LoD is defined as the minimum percentage of mutant DNA in a background of WT DNA

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that can be detected with a $95\%$ probability as determined by a probit analysis. A 5-level MAF% dilution series was made by serially diluting the original sample in a WT background. These dilution series were then tested using the therascreen PIK3CA RGQ PCR Kit for each of the mutations. The evaluation was performed using 3 different therascreen PIK3CA RGQ PCR Kit lots with 24 replicates tested per kit lot per level. The LoD value was determined as the highest value (in terms of MAF%) across all kit lots (Table 9).

Table 9: LoD for the 11 PIK3CA Mutations

| Mutation Name | LoD (MAF%) |
| --- | --- |
| C420R | 4.46* |
| E542K | 5.06† |
| E545A | 1.82* |
| E545D | 3.21* |
| E545G | 1.94† |
| E545K | 2.42† |
| H1047L | 2.37† |
| H1047R | 1.98† |
| H1047Y | 7.07* |
| Q546E | 5.31* |
| Q546R | 4.22* |

* LoD values were established using cell line samples
$\dagger$ LoD values were verified using intended use clinical plasma samples

# LoD confirmation with Clinical Samples

A combined precision and LoD study was performed such that the LoD for the therascreen PIK3CA RGQ PCR Kit was confirmed using clinical PIK3CA mutation positive and WT clinical plasma samples derived from the intended use population. Clinical samples, positive for the most prevalent mutations, H1047R, E545K, E542K and H1047L, were used in this study. A sample with a rare mutation (E545G) was also included in this study.

Extracted mutation positive DNA was normalized to a control assay Ct of 30.00 and diluted in clinical WT DNA to the MAF% equivalent to the estimated LoD. Prepared samples at LoD were tested using 2 lots of the therascreen PIK3CA PCR RGQ Kit with 2 operators, 1 run per operator per day for 2 non-consecutive days at 3 sites, with a total of 24 replicates (8 replicates per site).

The positive call rates for all variants were above $95\%$ , as shown in Table 10. Thus, the previously determined LoDs were confirmed for the tested mutations using clinical plasma samples.

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Table 10: LoD confirmation with clinical samples

| Mutation | Frequency of Positive Mutation Calls | Proportion of Positive Mutation Calls | Two-sided lower 95% CI | Two-sided lower 95% CI |
| --- | --- | --- | --- | --- |
| E542K | 24/24 | 100% | 85.75% | 100% |
| E545G | 23/24 | 95.83% | 78.88% | 99.89% |
| E545K | 24/24 | 100% | 85.75% | 100% |
| H1047L | 23/23 | 100% | 85.15% | 100% |
| H1047R | 21/22 | 95.45% | 77.16% | 99.88% |

# (iv) Effect of DNA Input on $\Delta C t$ (Linearity Studies)

The purpose of this study was to demonstrate that the performance of the therascreen PIK3CA RGQ PCR Kit is consistent across the DNA input range (control assay working range, 24.69 - 31.68 Ct); showing a consistent $\Delta$ Ct for a given MAF irrespective of the DNA input level. Genomic DNA (gDNA) was used for all 11 PIK3CA mutations in this study. The linear range of the therascreen PIK3CA RGQ PCR Kit was determined for each of the 11 mutation assays by testing across 8 DNA input levels with the upper level being outside of the control assay Ct working range (24.69 - 31.68 Ct). Each mutation sample was diluted to a constant MAF level representing 3X LoD and normalized to different target dilution control assay Ct values. The evaluation was performed using one therascreen PIK3CA RGQ PCR Kit lot with 2 replicates tested per DNA level.

The data was analyzed using regression analysis to determine the linear range. For the assay to be determined as linear across a DNA input, there should be no change across the range in $\Delta \mathrm{Ct}$ , i.e., there is no statistically significant linear, quadratic or cubic effect (i.e. $p &gt; 0.05$ ). A consistent linear $\Delta \mathrm{Ct}$ range was observed across the full tested DNA input range for E542K, E545K, E545D, E545A, H1047L, H1047Y, Q546R, C420R and H1047R assays. The E545G and Q546E assays did not show a linear $\Delta \mathrm{Ct}$ range across the full tested DNA input range. Removal of dilution 8 from the analysis of E545G and Q546E assays removed the non-linear effects such that the assays were linear across dilutions 1 to 7 which span the control assay working range. The linear range of the assays is shown below (Table 11).

Table 11: Linear Range of the Assay

| Assay | Linear Range based on observed Mean Control assay Ct |
| --- | --- |
| E542K | 25.21 to 33.35 |
| E545K | 24.98 to 33.45 |

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| Assay | Linear Range based on observed Mean Control assay Ct |
| --- | --- |
| E545D | 24.92 to 33.35 |
| E545G | 24.76 to 31.43 |
| E545A | 24.74 to 33.89 |
| H1047Y | 24.90 to 34.03 |
| Q546R | 24.76 to 32.12 |
| Q546E | 24.90 to 32.68 |
| C420R | 24.79 to 34.11 |
| H1047R | 25.00 to 33.41 |
| H1047L | 25.11 to 34.72 |

## 4. Analytical Specificity

### (i) Cross Reactivity and Primer and Probe Specificity

This study aimed to assess whether cross reactivity between mutations detected by the assay had been correctly accounted for in the setting of the analytical $\Delta C_t$ cut-off values of the therascreen PIK3CA RGQ PCR Kit. The cross reactivity of the six optimized reaction mixes was assessed by testing each tube against the 11 mutations detected by the therascreen PIK3CA RGQ PCR Kit. Samples were tested at low DNA input and low MAF% as well as high DNA input and high MAF%. Contrived plasma samples were used for all mutations. For each sample, two replicates were tested with 3 therascreen PIK3CA RGQ PCR Kit lots (generating 239 data points total). Overall, 1/239 (0.42%) data points showed mutant non-specific amplification. The single data point showing mutant non-specific (where a E542K low MAF sample was classified as Q546R) amplification was inconsistent with other replicates from the same sample, including the high DNA input, high MAF sample, which did not show the same mutant non-specific amplification. This result was therefore not considered to be a result of cross reactivity and was attributed to non-specific amplification.

Known PCR cross reactivity was observed during feasibility studies between H1047L and H1047R. This cross reactivity is uni-directional, i.e., if a double H1047L and H1047R sample is seen this will be reported as H1047R Mutation Detected. This rule is incorporated into the automated therascreen_PIK3CA_Plasma MDx Assay Profile algorithm.

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(ii) Cross Contamination

To evaluate the potential occurrence of cross contamination during the DNA extraction and subsequent *therascreen* PIK3CA RGQ PCR Kit testing procedures, this study was performed with H1047R (the highest prevalent mutation) and Wild Type contrived plasma samples. Samples were extracted following a pre-defined extraction matrix and mutation status was assessed across ten PCR runs. Eighteen extractions were carried out for the mutation positive (H1047R) samples and 42 extractions were carried out for the WT samples.

The observed percentage of correct mutation calls for Wild Type samples was 99.49%, (194 out of 195 replicates), demonstrating no significant cross contamination of the Wild Type samples by mutant samples sharing the same DNA extraction and run setup procedure.

(iii) Interfering Substances

The effect of potential interfering substances from both endogenous and exogenous substances was measured by comparison of $\Delta C_t$ between interferent spiked and control spiked extracts of each mutant (3X LoD) used within the study. The assessment of interfering substances for WT samples was made by evaluating all reaction mixes to demonstrate correct WT calls. Thirteen potential interfering substances were tested:

- Endogenous interfering substances that could be present in a clinical plasma sample: hemoglobin (2 g/L), triglycerides (37 mmol/L), EDTA (3.4 μmol/L), Caffeine (308 μmol/L), Albumin (30 mg/mL), Conjugated Bilirubin (342 μmol/L) and Unconjugated Bilirubin (342 μmol/L).
- Exogenous substances that could be introduced into a sample during the DNA extraction process: ethanol, proteinase K, buffer ACL, buffer ACB, buffer ACW1 and buffer ACW2, tested based on the highest carryover volume during extraction.

Contrived plasma samples were used for representative mutations for each reaction mix in the *therascreen* PIK3CA RGQ PCR Kit (E542K, E545K, H1047R, Q546R and C420R) and for WT sample. The evaluation was performed using one *therascreen* PIK3CA RGQ PCR Kit lot with 6 replicates tested per interferent. All results in both mutant and WT samples were as expected. Where a statistically significant difference was observed between spiked and control samples, this was within acceptable intermediate precision of the assay and was therefore within the inherent variability of the assay.

In a further analysis of the interfering substances data, the difference in absolute Ct for the mutant assays rather than $\Delta C_t$ was examined. Should a potential interferent have a proportionate effect on both the control and mutant Ct values (i.e., Ct values

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are shifted by a similar value due to the interferent), the resulting $\Delta C_t$ could remain comparable to the interferent free control despite a Ct shift. Therefore, this could mask the effect of the interferent which may result in a false negative call in samples close to the assay's LoD.

The acceptance criteria were kept consistent for the analysis of the absolute Ct values, that is for an interferent to be classed as having no impact, the p-value for the estimate should be $&gt;0.05$ or if the p-value $&lt; 0.05$ then the difference in absolute Ct must be within acceptable intermediate precision of the corresponding assay. The results showed that interferents tested had no impact on the absolute mutant Ct value. Based on the results, none of the interferents tested had any impact on kit performance.

# (iv) Repeatability and Reproducibility

The objective of this study was to demonstrate the repeatability and reproducibility of the therascreen PIK3CA RGQ PCR Kit (including intermediate precision).

The reproducibility of the therascreen PIK3CA RGQ PCR Kit for the detection of mutations in the PIK3CA gene was evaluated in two studies. The first study used WT and 11 contrived cell line DNA samples carrying the PIK3CA mutations spiked into HD plasma. All extractions were carried out using two lots of QIAamp DSP Circulating Nucleic Acid Kit. Extracted DNA was normalized to a control assay Ct value of 30 and diluted to the MAF levels of 1X LoD and 3X LoD.

To demonstrate repeatability, two runs per day, by three operators and using three instruments, were carried out across 20 non-consecutive days to give a total of 120 runs at Site 1. The proportion of correct mutation calls and the lower one-sided exact $95\%$ confidence intervals (95% CI) were reported in Table 12 below.

Table 12: Repeatability - Proportion of Correct Calls for PIK3CA Mutation Tested

| Mutation | Template | Fractional Proportion | Percentage | Two-Sided Lower 95% Confidence Limit |
| --- | --- | --- | --- | --- |
| C402R | LOD | 120/120 | 100.00% | 96.97% |
| | 3xLOD | 120/120 | 100.00% | 96.97% |
| E542K | LOD | 120/120 | 100.00% | 96.97% |
| | 3xLOD | 120/120 | 100.00% | 96.97% |
| E542A | LOD | 119/120 | 99.17% | 95.44% |
| | 3xLOD | 120/120 | 100.00% | 96.97% |
| E545D | LOD | 120/120 | 100.00% | 96.97% |
| | 3xLOD | 120/120 | 100.00% | 96.97% |
| E545G | LOD | 119/120 | 99.17% | 95.44% |
| | 3xLOD | 120/120 | 100.00% | 96.97% |

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| Mutation | Template | Fractional Proportion | Percentage | Two-Sided Lower 95% Confidence Limit |
| --- | --- | --- | --- | --- |
| E545K | LOD | 111/120 | 92.50% | 86.24% |
| | LOD* | 120/120 | 100.00% | 96.97% |
| H1047L | LOD | 120/120 | 100.00% | 96.97% |
| | 3xLOD | 120/120 | 100.00% | 96.97% |
| H1047R | LOD* | 110/120 | 91.67% | 85.21% |
| | 3xLOD* | 120/120 | 100.00% | 96.97% |
| H1047Y | LOD | 120/120 | 100.00% | 96.97% |
| | 3xLOD | 120/120 | 100.00% | 96.97% |
| Q546E | LOD | 120/120 | 100.00% | 96.97% |
| | 3xLOD | 120/120 | 100.00% | 96.97% |
| Q546R | LOD | 115/120 | 95.83% | 90.54% |
| | 3xLOD | 120/120 | 100.00% | 96.97% |
| WT | Ct30 | 114/120 | 95.00% | 89.43% |

*For E545K and H1047R the LoD used were 1.99 and 1.44, respectively. The LoD was readjusted and confirmed in a subsequent study. The readjusted LoD was used in the subsequent study (see Table 13 below).

Reproducibility was assessed over three sites Site 1, Site 2 and Site 3 (Table 13). Two runs a day were performed per three operators and three instruments per site by the two external sites (Sites 2 and 3) over 10 non-consecutive days to give an additional 60 runs per external site. Three unique lots of reagents were included in the study where each lot was tested at two sites. Three sets of single use aliquots were prepared for each of the samples; one set was kept at Site 1 for testing while the remaining two sets and were shipped, on dry ice, to the two external sites for testing.

Proportion of correct calls with two-sided lower $95\%$ confidence level and variance component analysis in terms of standard deviation (SD) of $\Delta C_t$ are included in Table 14 and Table 15.

Table 13: Reproducibility - Proportion of Correct Calls for PIK3CA Mutations Tested Across All Sites

| Mutation | Template | Fractional Proportion of Valid Results | Percentage | Two-Sided Lower 95% Confidence Limit |
| --- | --- | --- | --- | --- |
| C420R | LoD | 237/238 | 99.58% | 97.68% |
| | 3x LoD | 238/238 | 100.00% | 98.46% |
| E542K | LOD | 237/240 | 98.75% | 96.39% |
| | 3xLOD | 240/240 | 100.00% | 98.47% |
| E545A | LOD | 239/240 | 99.58% | 97.70% |

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| Mutation | Template | Fractional Proportion of Valid Results | Percentage | Two-Sided Lower 95% Confidence Limit |
| --- | --- | --- | --- | --- |
| | 3xLOD | 240/240 | 100.00% | 98.47% |
| E545D | LOD | 240/240 | 100.00% | 98.47% |
| | 3xLOD | 240/240 | 100.00% | 98.47% |
| E545G | LOD | 237/240 | 98.75% | 96.39% |
| | 3xLOD | 239/239 | 100.00% | 98.47% |
| E545K | LOD* | 432/432 | 100.00% | 99.15 |
| | 3xLOD | 240/240 | 10.000% | 89.47% |
| H1047L | LOD | 236/238 | 99.16% | 97.00% |
| | 3xLOD | 238/238 | 100.00% | 98.46% |
| H1047R | LOD* | 430/432 | 99.54% | 98.34% |
| | 3xLOD | 236/236 | 100.00% | 98.45% |
| H1047Y | LOD | 239/240 | 99.58% | 97.70% |
| | 3xLOD | 240/240 | 100.00% | 98.47% |
| Q546E | LOD | 238/238 | 100.00% | 98.46% |
| | 3xLOD | 238/238 | 100.00% | 98.46% |
| Q546R | LOD | 232/240 | 96.67% | 93.54% |
| | 3xLOD | 240/240 | 100.00% | 98.47% |
| WT | Ct30 | 223/238 | 93.70 | 89.82 |

*Samples at revised LoD with E545K and H1047R (as per Table 6) were evaluated for 6 days across 3 sites, by 3 operators, with 2 runs and 4 replicates for a total of 144 measurements per site, 432 total in 3 sites.

All mutant samples demonstrated 100% agreement at 3X LoD. In addition, at 1X LoD, all 11 mutants showed &gt;99% agreement except Q546R at 1X LoD.

In the repeatability study the WT sample correct call rate was 95.00% (data not shown), with a lower two-sided exact 95% confidence limit of 89.43%. Similarly, in the reproducibility study, the percentage correct calls for the WT samples was 93.70% (data not shown), resulting in a lower two-sided 95% confidence limit of 89.82%. An additional Reproducibility and Repeatability will be performed to evaluate WT and mutation positive samples per the Instructions for Use, that is, by testing all 6 reactions mixes per sample (see CDRH decision below).

The second reproducibility study includes the LoD confirmation study with 5 clinical samples which is described in Section 3 (iv), above.

The WT sample evaluated in the repeatability and reproducibility study did not meet the acceptance criteria in relation to proportion of correct calls. An investigation indicated that the WT sample had an additional mutation with low allelic frequency

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as determined by an NGS-based method. A new repeatability and reproducibility study will be conducted postmarket with an appropriately qualified WT sample. Refer to section XIV.

A variance component analysis was used to estimate the standard deviation for between-run, between-day, between-lot, between-operator, between-instrument, and between-day variability for the reproducibility study (Table 14).

Table 14: Repeatability - Standard Deviation of Variance Components

| Mutation | Level | Number | Mean | Between Kit Lot SD | Between Run SD | Between Operator SD | Between Day SD | Between Instrument SD | Within Run SD | Total SD |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| E542K | 3xLOD | 120 | 1.06 | 0.09 | 0.18 | 0.07 | 0.00 | 0.21 | 0.21 | 0.33 |
| | LOD | 120 | 3.28 | 0.02 | 0.10 | 0.00 | 0.00 | 0.24 | 0.46 | 0.51 |
| E545D | 3xLOD | 120 | 2.97 | 0.000 | 0.10 | 0.00 | 0.000 | 0.07 | 0.30 | 0.32 |
| | LOD | 120 | 4.1 | 0.09 | 0.00 | 0.00 | 0.12 | 0.07 | 0.48 | 0.50 |
| E545G | 3xLOD | 120 | 1.9 | 0.01 | 0.17 | 0.00 | 0.00 | 0.00 | 0.32 | 0.36 |
| | LOD | 120 | 5.72 | 0.00 | 0.24 | 0.00 | 0.00 | 0.27 | 1.06 | 1.1 |
| E545K | 3xLOD | 120 | 4.57 | 0.09 | 0.07 | 0.00 | 0.00 | 0.22 | 0.58 | 0.62 |
| | LOD | 144 | 5.28 | 0.13 | 0.086 | 0.19 | 0.00 | 0.42 | 0.66 | 0.77 |
| E545A | 3xLOD | 120 | 0.85 | 0.05 | 0.17 | 0.00 | 0.00 | 0.05 | 0.30 | 0.35 |
| | LOD | 120 | 3.66 | 0.00 | 0.37 | 0.00 | 0.40 | 0.26 | 1.21 | 1.3 |
| H1047Y | 3xLOD | 120 | 3.45 | 0.06 | 0.12 | 0.02 | 0.00 | 0.09 | 0.25 | 0.29 |
| | LOD | 120 | 5.05 | 0.00 | 0.00 | 0.02 | 0.00 | 0.13 | 0.41 | 0.43 |
| Q546R | 3xLOD | 120 | 1.91 | 0.057 | 0.26 | 0.00 | 0.00 | 0.00 | 0.36 | 0.44 |
| | LOD | 120 | 4.74 | 0.29 | 0.11 | 0.16 | 0.34 | 0.180 | 1.07 | 1.16 |
| C420R | 3xLOD | 120 | 1.45 | 0.04 | 0.06 | 0.04 | 0.08 | 0.20 | 0.30 | 0.36 |
| | LOD | 120 | 3.23 | 0.000 | 0.09 | 0.00 | 0.21 | 0.18 | 0.65 | 0.71 |
| Q546E | 3xLOD | 120 | 1.14 | 0.06 | 0.11 | 0.00 | 0.00 | 0.00 | 0.21 | 0.24 |

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| Mutation | Level | Number | Mean | Between Kit Lot SD | Between Run SD | Between Operator SD | Between Day SD | Between Instrument SD | Within Run SD | Total SD |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | LOD | 120 | 5.38 | 0.21 | 0.00 | 0.123 | 0.21 | 0.17 | 0.83 | 0.88 |
| H1047L | 3xLOD | 120 | 2.18 | 0.00 | 0.14 | 0.03 | 0.10 | 0.11 | 0.22 | 0.30 |
| | LOD | 120 | 5.18 | 0.20 | 0.00 | 0.00 | 0.00 | 0.00 | 0.63 | 0.64 |
| H1047R | 3xLOD | 120 | 3.68 | 0.07 | 0.19 | 0.03 | 0.06 | 0.03 | 0.52 | 0.56 |
| | LOD | 143 | 4.49 | 0.00 | 0.20 | 0.00 | 0.00 | 0.16 | 0.75 | 0.77 |

Table 15: Reproducibility - Standard Deviation of Variance Components

| Mutation | Level | Nimber | Mean | Between Site (SD) | Between Run Within Site (SD) | Between Operator (SD) | Between Instrument (SD) | Between Day (SD) | Between Lot (SD) | Within Site (SD) | Within Run (SD) | Total (SD) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| E542K | 3xLOD | 240 | 1.07 | 0.14 | 0.20 | 0.03 | 0.25 | 0.00 | 0.06 | 0.41 | 0.24 | 0.40 |
| | LOD | 240 | 3.24 | 0.13 | 0.24 | 0.00 | 0.32 | 0.00 | 0.00 | 0.63 | 0.48 | 0.61 |
| E545D | 3xLOD | 240 | 3.00 | 0.08 | 0.16 | 0.00 | 0.09 | 0.00 | 0.00 | 0.37 | 0.32 | 0.40 |
| | LOD | 240 | 4.14 | 0.11 | 0.12 | 0.00 | 0.17 | 0.00 | 0.00 | 0.55 | 0.51 | 0.55 |
| E545G | 3xLOD | 239 | 1.88 | 0.00 | 0.08 | 0.00 | 0.00 | 0.00 | 0.00 | 0.36 | 0.35 | 0.36 |
| | LOD | 239 | 5.82 | 0.39 | 0.10 | 0.00 | 0.26 | 0.16 | 0.14 | 1.16 | 1.10 | 1.18 |
| E545K | 3xLOD | 240 | 4.74 | 0.19 | 0.00 | 0.00 | 0.29 | 0.00 | 0.00 | 0.72 | 0.66 | 0.72 |
| E545K | LOD | 432 | 4.99 | 0.34 | 0.21 | 0.10 | 0.28 | 0.13 | 0.07 | 0.75 | 0.63 | 0.79 |
| E545A | 3xLOD | 240 | 0.91 | 0.00 | 0.12 | 0.01 | 0.11 | 0.00 | 0.00 | 0.39 | 0.35 | 0.39 |
| | LOD | 240 | 3.67 | 0.00 | 0.34 | 0.00 | 0.19 | 0.07 | 0.00 | 1.18 | 1.09 | 1.18 |

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| Mutation | Level | Number | Mean | Between Site (SD) | Between Run Within Site (SD) | Between Operator (SD) | Between Instrument (SD) | Between Day (SD) | Between Lot (SD) | Within Site (SD) | Within Run (SD) | Total (SD) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| H1047Y | 3xLOD | 240 | 3.42 | 0.08 | 0.13 | 0.00 | 0.14 | 0.00 | 0.07 | 0.32 | 0.25 | 0.32 |
| | LOD | 240 | 5.02 | 0.02 | 0.05 | 0.00 | 0.15 | 0.00 | 0.01 | 0.44 | 0.41 | 0.44 |
| Q546R | 3xLOD | 240 | 1.87 | 0.06 | 0.22 | 0.00 | 0.03 | 0.00 | 0.03 | 0.44 | 0.38 | 0.44 |
| | LOD | 240 | 4.66 | 0.10 | 0.16 | 0.16 | 0.35 | 0.00 | 0.18 | 1.15 | 1.06 | 1.13 |
| C420R | 3xLOD | 238 | 1.36 | 0.06 | 0.12 | 0.00 | 0.17 | 0.04 | 0.05 | 0.38 | 0.31 | 0.38 |
| | LOD | 238 | 3.18 | 0.00 | 0.19 | 0.00 | 0.12 | 0.11 | 0.05 | 0.70 | 0.65 | 0.70 |
| Q546E | 3xLOD | 238 | 1.11 | 0.00 | 0.08 | 0.00 | 0.00 | 0.03 | 0.07 | 0.25 | 0.22 | 0.24 |
| | LOD | 238 | 5.36 | 0.00 | 0.17 | 0.00 | 0.07 | 0.12 | 0.00 | 0.90 | 0.87 | 0.90 |
| H1047L | 3xLOD | 238 | 2.08 | 0.08 | 0.14 | 0.02 | 0.09 | 0.02 | 0.10 | 0.30 | 0.23 | 0.30 |
| H1047L | LOD | 238 | 5.18 | 0.00 | 0.00 | 0.13 | 0.00 | 0.11 | 0.00 | 0.99 | 0.97 | 0.99 |
| H1047R | 3xLOD | 236 | 3.67 | 0.00 | 0.18 | 0.00 | 0.10 | 0.00 | 0.00 | 0.64 | 0.61 | 0.64 |
| H1047R | LOD | 430 | 4.41 | 0.17 | 0.21 | 0.00 | 0.08 | 0.07 | 0.09 | 0.82 | 0.77 | 0.82 |

(v) Lot-to-Lot Reproducibility

To demonstrate lot-to-lot interchangeability, three lots of the Plasma Extraction Kit and one lot of the therascreen PIK3CA RGQ PCR Kit were used to test contrived plasma samples for all 11 mutations and HD plasma as the WT sample. Each sample was extracted with three different lots of the QIAamp DSP Circulating Nucleic Acid Kit. The ages of the three different DNA Extraction Kit lots used for the lot-to-lot study were 21.6 months, 10.5 months, and 9.6 months. Six replicate extractions were carried out per sample per extraction kit lot. The correct overall percentage mutation result was reported across all samples and kit lots, along with the corresponding two-sided exact 95% confidence limits. For each therascreen PIK3CA RGQ PCR Kit lot and Plasma Extraction Kit lot combination, a total of 216 valid data points (12 targets x 6 replicates x 3 extraction kit lots) were collected. The percentage of correct overall mutation call was 100% (216/216) across all samples (11 mutants and the wild type) and extraction kit lots, with the corresponding two

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sided exact 95% confidence limits of 98.31% and 100%. Additionally no statistically significant difference was observed in ΔCt values for any mutation positive samples between extraction kits lots.

(vi) Sample Handling

Patient samples were collected at the SOLAR-1 drug investigational sites in accordance with the SOLAR-1 protocol and supporting documents. Patient blood samples were collected at cycle one day one after enrollment into the trial and prior to start of SOLAR-1 study treatment. Blood was collected in 10 mL EDTA tubes and within 2 hours plasma was separated by double centrifugation (10 minutes at room temperature at 1,600 g and 3,000 g, respectively). The plasma was aliquoted at approximately 1 mL into 2 mL plain cap pre-labeled cryogenic vials and stored at -70°C or colder.

The frozen plasma samples were shipped on dry ice to for central storage at -70°C or colder. Prior to testing, frozen samples were shipped on dry ice from the central storage to the sample testing site.. At the testing site, frozen samples were received and stored at -70°C to -90°C until further testing.

To demonstrate that different laboratories produce acceptable results when starting from the same plasma sample, extractions were performed across 3 different sites. Contrived samples consisting of fragmented cell line DNA spiked into HD plasma for all 11 mutations as well as a WT sample were used in this study. There were 18 × 2 mL aliquots required for each sample; these aliquots were randomized and split into 18 extract sets. These extract sets were then distributed evenly across the three testing sites, six extracts per study site. Testing of the extracts using the therascreen PIK3CA RGQ PCR Kit was performed at Site 1. When comparing the results of each sample across all 3 sites, the percentage of correct mutation calls for mutation positive and WT samples was 100%.

(vii) Guardbanding

Four guardbanding studies (PCR annealing temperature, Reagent volume, Thawing and PCR Set-Up) were designed to assess the performance of the PCR kit which is shared by both the Tissue and Plasma indications (see P190001 SSED),

A guardband study was conducted for the QIAamp DSP Circulating Nucleic Acid Kit (specific for Plasma) using WT and mutation positive contrived plasma samples for E542K, E545K, Q546R, C420R and H1047R) that could be detected by each of the reaction mixes within the kit normalized to a Ct value of 30 and diluted to a MAF% of 3X LoD. MAF% dilutions were prepared in a background of WT DNA. DNA was extracted using QIAamp DSP Circulating Nucleic Acid Kit. During the extraction process the following conditions were varied:

- Proteinase K incubation time (Time 1, 30min at standard conditions ±10min) and temperature (Temp. 1, 60°C at standard conditions ±5 °C)

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- Membrane drying time (Time 2, 10min at standard conditions ±5min) and temperature (Temp. 2, 56°C at standard conditions ±5 °C)

Twenty unique combinations of the varied conditions including standard conditions were tested so that the main and second order polynomial effects could be estimated for all possible combinations. To determine the effect of varying incubation temperature (Incubation 1: 55°C to 65°C, Incubation 2: 51°C to 61°C) and incubation time (Incubation 1: 20 mins to 40 mins, Incubation 2: 5 mins to 15 mins) during the DNA extraction process, the temperature and duration of two incubations were varied while keeping all other factors constant.

For WT samples the correct overall mutation call was determined across all tested conditions; 100% correct mutation call rate ("No mutation detected") was observed. Therefore, varying the incubation time and temperature from the standard condition had no impact on the WT correct call rate.

For each mutation positive sample, separate ANOVA models were fitted with ΔCt as the response variable, centralized (to the standard condition) incubation temperatures and times along with their corresponding second order interactions and power terms as continuous explanatory variables. For each of the mutant samples, the estimated differences to the nominal guardband condition were generated.

The acceptable extraction time and temperature tolerances, which have been demonstrated to have no significant impact on the performance of the therascreen PIK3CA RGQ PCR Kit are shown in Table 16.

Table 16: Acceptable Plasma Extraction Parameters
| Step | Description | Handbook condition | Acceptable variation |
| --- | --- | --- | --- |
| 8 (Temp 1) | Incubation with Proteinase K | 60 °C | ±2°C |
| 8 (Time 1) | | 30 min | ±4 mins |
| 24 (Temp 2) | Drying Membrane | 56 °C | ±3°C |
| 24 (Time 2) | | 10 min | ±4 mins |

Correct mutation call was observed for all WT and mutation positive samples under all conditions tested. Acceptable time and temperature tolerances were established.

(viii) Blood Collection Tube Validation

This study was designed to determine the impact of blood to plasma separation time on plasma sample quality and subsequent results when tested with the therascreen

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PIK3CA RGQ PCR Kit. The time up to four hours between blood collection and plasma separation was assessed.

Contrived blood samples for H1047R (the most prevalent mutation) and whole blood samples as WT samples were used in this study. Blood samples were collected into 10 mL K₂EDTA tubes from four donors (eight tubes per donor). Contrived blood samples were generated by spiking H1047R fragmented cell line DNA into blood tubes from two donors after collection. Blood samples were separated into plasma at 1h, 2h, 3h and 4h timepoints post collection. DNA was extracted from the plasma samples using the QIAamp DSP Circulating Nucleic Acid Kit and each target was tested with the therascreen PIK3CA RGQ PCR Kit in 16 replicates per each timepoint.

The percentage of correct calls based on the overall mutation status (i.e. Mutation Detected or No Mutation Detected) was 100% for all samples across all donors at each timepoint. There was no statistically significant drift in ΔCt observed for the H1047R sample. This study demonstrated that there is no impact of the blood to plasma separation time, if processed within four hours, on the therascreen PIK3CA RGQ PCR Kit.

(ix) Stability Studies

(a) Stability of Specimens (Plasma)

Long term stability of two mutant positive clinical plasma samples (E545K and H1047R) is currently being assessed when stored at -80°C. Samples will be extracted and tested with the therascreen PIK3CA RGQ PCR Kit at the following time points: baseline (T0), 7 months (T1), 12 months (T2), 19 months (T3), 24 months (T4) and 25 months (T5).

So far the interim stability data includes time points T0, T1 and T2 (up to 12 months). Three clinical samples have been used in this study; E545K and H1047R mutation positive samples (around 2X LoD) in addition to a WT plasma sample. A 100% correct call rate was achieved across all samples at all time points (6 replicates per sample per time point). The data has demonstrated that plasma samples are stable when stored at -80°C for ≤ 11 months.

Stability of plasma samples stored at -80°C will be tested over a 25 month time frame to assess long term storage.

Plasma sample stability in simulated shipping conditions have also been assessed up to eight days using clinical mutation positive and WT samples stored on dry ice. 100% correct call rate was achieved at the tested time points demonstrating plasma samples are stable when shipped on dry ice for ≤ 7 days.

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(b) Stability of Specimens (DNA)

One clinical sample and five contrived plasma samples, generated by spiking fragmented cell line DNA into HD plasma, for E542K, E545K, Q546R, C420R and H1047R were used in this study. DNA from contrived plasma samples was extracted using the QIAamp DSP Circulating Nucleic Acid Kit and DNA eluates were normalized to the control assay Ct of 30. All contrived samples used in this study were normalized to an MAF level corresponding to 1.5X LoD. The clinical sample was adjusted to 3X LoD.

To assess the stability of extracted DNA when stored at -35°C to -15°C for the therascreen PIK3CA RGQ PCR Kit, the stability of DNA extracted was tested (6 replicates per sample per time point) against temporal and freeze-thaw conditions:

- DNA was stored at -15°C to -35°C for 2, 4 and 5 weeks before testing with the therascreen PIK3CA RGQ PCR Kit
- DNA was subjected to 1 – 5 freeze-thaw cycles over the course of five weeks before testing with the therascreen PIK3CA RGQ PCR Kit. Samples were stored at -15°C to -35°C between freeze-thaw cycles.

All tested samples at all time points were valid and the correct call rate was 100% in both temporal and freeze-thaw cycle stability studies. Results from this study show that DNA extracted from plasma specimens can be stored at -20°C (range: -35 to -15°C) for up to four weeks and undergo up to four freeze-thaw cycles.

(c) Kit Stability

The kit stability study for both tissue and plasma was done simultaneously using the same protocol with the exception that the PIK3CA mutant used for the study was different. For the plasma test, E545K was used and for tissue, H1047R was used.

The therascreen PIK3CA RGQ PCR Kit was assessed for:

- Real-Time stability (closed bottle, post-transport simulation)
- In-Use stability (including freeze-thaw and open vial, post-transport simulation)
- Transport simulation stability (integrated in real-time and in-use stability study)

To evaluate real-time and in-use stability all test time points assessed the same batch of extracted plasma DNA from mutant (E545K) positive clinical samples

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at 2X LoD and WT, as well as cell line mutant sample for E545K at 3X LoD level. Transport Simulation and In-Use stability were assessed using one therascreen PIK3CA RGQ PCR Kit. Real-Time stability was assessed using three therascreen PIK3CA RGQ PCR Kit lots. Six replicates were tested at each time point for Real-Time stability. Two replicates were tested at each time point for In-Use stability. At time point 13 months, 100% correct calls were observed for all samples and test conditions tested (Real-Time, In-Use and Transport stability). A linear regression for ΔCt values (for the Cell Line H1047R mutation positive sample) also showed no significant shift in ΔCt over the time tested.

The real time stability data generated during this study supports a shelf-life claim of 12 months the therascreen PIK3CA RGQ PCR Kit when stored at -30°C to -15°C.

The in-use data generated during this study for the therascreen PIK3CA RGQ PCR Kit at all time points up to 13 months confirms the stability after 5 freeze-thaw cycles.

A post market study will be conducted using representative mutation positive samples from each reaction tube at 2X LoD to validate and support the reagent stability of all the components of the therascreen PIK3CA RGQ PCR Kit. Refer for section XIV.

(d) Plasma DNA Extraction Kit Stability

The preliminary stability of the QIAamp® DSP Circulating Nucleic Acid Kit was provided to allow a minimum stability claim for 6 months. A post market study based on an approved protocol will be conducted using representative mutation positive to demonstrate real-time stability of the QIAamp® DSP Circulating Nucleic Acid Kit. Refer for section XIV.

B. Animal Studies

None

C. Additional Studies

None

X. SUMMARY OF PRIMARY CLINICAL STUDY

The QIAGEN GmbH (QIAGEN) performed a clinical performance study to establish a reasonable assurance of safety and effectiveness of the therascreen PIK3CA RGQ PCR Kit as an aid to clinicians in identifying breast cancer patients who may be eligible for treatment with PIQRAY® (alpelisib), based on a PIK3CA mutation detected result in plasma specimens. The study was originally initiated under Investigational Device Exemption (IDE) G160122 when the patients were selected and randomized based on the results from the patients tissue specimens using a Clinical Trial Assay (CTA) and the therascreen

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PIK3CA RGQ PCR Kit. The results from the concordance study between the tissue and plasma test, and the clinical efficacy from the clinical study were the basis for the PMA approval decision. A summary of the clinical study is presented below.

Clinical performance of the Tissue test using the therascreen PIK3CA RGQ PCR Kit (PIK3CA tissue) was demonstrated (see SSED for P190001) using tumor biopsies from patients enrolled in study CBYL719C2301 (SOLAR-1). SOLAR-1 is a pivotal registrational trial for alpelisib, an α-specific phosphatidylinositol-3-kinase (PI3K) inhibitor. For detailed description of SOLAR-1 study design, see SSED for P190001.

A summary of the study conducted to support an alpelisib selection claim is presented below.

## A. Study Design

For a complete SOLAR-1 study design see SSED for P190001. The study was initiated on July 23, 2015. The cut-off date for efficacy analysis in PIK3CA mutant cohort and safety for both cohorts is June 12, 2018. The database for this PMA reflected data collected through June 12, 2018 for the PIK3CA mutant cohort and December 23, 2016 for the PIK3CA non-mutant cohort and included 572 patients.

The study was a pivotal Phase III, randomized, double-blind, placebo controlled, international, multicenter study of alpelisib in combination with fulvestrant in men and postmenopausal women with hormone receptor positive (HR+), human epidermal growth factor receptor 2 negative (HER2-) locally advanced breast cancer whose disease had progressed or recurred on or after an aromatase inhibitor based treatment (with or without CDK4/6 combination).

A total of 572 breast cancer patients were enrolled into two cohorts, with or without a PIK3CA mutation. Patients and investigators were blinded to mutational status. Patients were randomized to receive either alpelisib 300 mg plus fulvestrant or placebo plus fulvestrant in a 1:1 ratio. Randomization was stratified by presence of lung and/or liver metastasis and previous treatment with Cyclin-Dependent Kinases 4 and 6 (CDK4/6) inhibitor(s). Blood samples for plasma ctDNA were collected from randomized patients on Cycle 1 Day 1 prior to initiation of study treatment.

During the randomized treatment phase, alpelisib 300 mg or placebo was administered orally once daily on a continuous basis. Fulvestrant 500 mg was administered intramuscularly on Cycle 1 Day 1 and 15 and then at Day 1 of a 28-day cycle during treatment phase (administration +/- 3 days). Patients were not allowed to cross over from placebo to alpelisib during the study or after disease progression.

The primary endpoint for the SOLAR-1 clinical study was progression free survival (PFS) using Response Evaluation Criteria in Solid Tumors (RECIST v1.1), based on investigator assessment in advanced breast cancer patients enrolled with a PIK3CA mutation as determined in FFPE tumor tissue. A secondary endpoint was PFS for

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patients without PIK3CA mutation as determined in tumor tissue, and PFS for patients with and without PIK3CA mutations as determined in plasma at baseline.

For the plasma testing, the plasma samples collected prior to study treatment were retrospectively tested to determine PIK3CA mutation status using the therascreen PIK3CA RGQ PCR Kit (hereafter refer to as therascreen PIK3CA RGQ PCR Kit using plasma). The patients had been previously tested for PIK3CA mutation status with the therascreen PIK3CA RGQ PCR Kit using tissue biopsy samples. Clinical efficacy of alpelisib in combination with fulvestrant was estimated for patients with a PIK3CA mutation as determined using plasma samples. Agreement of PIK3CA mutation results from plasma samples was compared to results from tissue samples for the SOLAR-1 randomized population. Secondary analyses were performed to estimate efficacy in the plasma negative population and in the plasma/tissue subpopulations.

Informed consent for the use of blood samples was obtained from patients in accordance with the Novartis CBYL719C2301 clinical protocol.

## 1. Key Inclusion and Exclusion Criteria

Enrollment in the SOLAR-1 study was limited to patients who met the following key inclusion criteria:

- Adults (female postmenopausal or men) ≥ 18 years old
- Radiological or objective evidence of recurrence or
- progression during or after Aromatase Inhibitor (AI) therapy
- Histologically and/or cytologically confirmed diagnosis of estrogen receptors (ER)-positive and/or Progesterone receptor positive breast cancer
- Has HER2- breast cancer
- Identified PIK3CA status (mutant or non-mutant; determined by a Novartis designated laboratory).
- Subject either had
- Measurable disease, i.e. at least one measurable lesion per RECIST 1.1 or
- If no measurable disease was present, then at least one predominantly lytic bone lesion must be present
- Subject had advanced (locoregionally recurrent not amenable to curative therapy or metastatic) breast cancer
- Eastern Cooperative Oncology Group (ECOG) performance status 0 or 1
- Adequate bone marrow and organ function as described in protocol

Patients were not permitted to enroll in the SOLAR-1 study if they met any of the following key exclusion criteria:

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- Had inflammatory breast cancer or uncontrolled central nervous system metastases.
- Had concurrent malignancy or malignancy within 3 years of randomization, except for adequately treated, basal or squamous cell carcinoma, non-melanomatous skin cancer, or curatively resected cervical cancer.
- Received prior treatment with chemotherapy in the advanced setting or prior therapy with fulvestrant or any PI3K, AKT or mTOR inhibitor
- Had Type 1 or uncontrolled Type 2 diabetes mellitus
- Had documented pneumonitis at time of screening

## 2. Follow-up Schedule

### Safety follow-up

Subjects who discontinued study treatment were followed for safety, until 30 days after last study treatment administration, except in the case of death, loss to follow-up, or withdrawal of consent.

### Efficacy follow-up

Subjects who discontinued study treatment for reasons other than disease progression, death, loss to follow-up, or withdrawal of consent, were followed every eight weeks ± one week for efficacy during the first 18 months and every 12 weeks ± one week thereafter until disease progression, death, loss to follow-up, or withdrawal of consent.

### Survival follow-up

All subjects were followed for survival every 12 weeks until death, loss to follow-up or withdrawal of consent until the final number of OS events was reached or if the study had stopped for other reasons. During the survival follow-up, data relaed to subsequent antineoplastic therapies initiated after study treatment discontinuation were collected along with the start/end date and date of disease progression on subsequent therapies.

## 1. Clinical Endpoints

### Primary Efficacy Endpoint: PFS in PIK3CA mutant cohort

The primary endpoint for the study was PFS using RECIST v1.1 criteria, based on investigator assessment in advanced breast cancer patients enrolled with a PIK3CA mutation based on tissue tumor samples.

### Safety Endpoints

Safety and tolerability were evaluated by assessment of type, frequency and severity of adverse events and laboratory toxicities per Common Terminology Criteria for Adverse Events (CTCAE) v4.03.

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## 2. Plasma Diagnostic Study Objective and Design

The primary objectives of the study using Plasma specimens were

- to demonstrate the clinical utility of the therascreen PIK3CA RGQ PCR Kit using plasma in identifying breast cancer patients with a PIK3CA mutation for treatment with alpelisib in combination with fulvestrant, and
- to evaluate the concordance of PIK3CA mutation status in plasma compared to tissue samples.

The secondary objectives of the therascreen PIK3CA RGQ PCR Kit using plasma were

- to evaluate the clinical efficacy of PIK3CA mutation negative patients identified by the therascreen PIK3CA RGQ PCR Kit for tr…